Compositions and methods for locating preferred integration sites within the genome of a plant

ABSTRACT

Methods to find optimal integration sites within a plant genome are provided. More particularly, a plant is transformed with a target site having an expression cassette comprising a nucleotide sequence operably linked to a promoter active in the plant. The target site is flanked by non-identical recombination sites. Transformed protoplast, tissues, or whole plants can be tested to determine the levels of activity of the inserted gene. By comparison of cellular activities of the gene in different insertion sites, preferred integration sites may be found wherein the gene is expressed at high or acceptable levels. These plants can then be utilized with subsequent retargeting techniques to replace the nucleotide sequence with other genes or nucleotide sequences of interest contained in a transfer cassette.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No.09/455,050, filed Dec. 6, 1999, which is a divisional application ofU.S. application Ser. No. 09/193,502 filed Nov. 17, 1998, now U.S. Pat.No. 6,187,994, which claims the benefit of U.S. Provisional ApplicationSerial No. 60/065,627, filed Nov. 18, 1997, and U.S. Application SerialNo. 60/065,613, filed Nov. 18, 1997, all of which are hereinincorporated by reference.

FIELD OF THE INVENTION

[0002] The invention relates to the genetic modification of plants.Particularly, the control of gene integration and expression in plantsis provided.

BACKGROUND OF THE INVENTION

[0003] Genetic modification techniques enable one to insert exogenousnucleotide sequences into an organism's genome. A number of methods havebeen described for the genetic modification of plants. All of thesemethods are based on introducing a foreign DNA into the plant cell,isolation of those cells containing the foreign DNA integrated into thegenome, followed by subsequent regeneration of a whole plant.Unfortunately, such methods produce transformed cells that contain theintroduced foreign DNA inserted randomly throughout the genome and oftenin multiple copies.

[0004] The random insertion of introduced DNA into the genome of hostcells can be lethal if the foreign DNA happens to insert into, and thusmutate, a critically important native gene. In addition, even if arandom insertion event does not impair the functioning of a host cellgene, the expression of an inserted foreign gene may be influenced by“position effects” caused by the surrounding genomic DNA. In some cases,the gene is inserted into sites where the position effects are strongenough to prevent the synthesis of an effective amount of product fromthe introduced gene. In other instances, overproduction of the geneproduct has deleterious effects on the cell.

[0005] Transgene expression is typically governed by the sequences,including promoters and enhancers, which are physically linked to thetransgene. Currently, it is not possible to precisely modify thestructure of transgenes once they have been introduced into plant cells.In many applications of transgene technology, it would be desirable tointroduce the transgene in one form, and then be able to modify thetransgene in a defined manner. By this means, transgenes could beactivated or inactivated where the sequences that control transgeneexpression can be altered by either removing sequences present in theoriginal transgene or by inserting additional sequences into thetransgene.

[0006] For higher eukaryotes, homologous recombination is an essentialevent participating in processes like DNA repair and chromatid exchangeduring mitosis and meiosis. Recombination depends on two highlyhomologous extended sequences and several auxiliary proteins. Strandseparation can occur at any point between the regions of homology,although particular sequences may influence efficiency. These processescan be exploited for a targeted integration of transgenes into thegenome of certain cell types.

[0007] Even with the advances in genetic modification of higher plants,the major problems associated with the conventional gene transformationtechniques have remained essentially unresolved as to the problemsdiscussed above relating to variable expression levels due tochromosomal position effects and copy number variation of transferredgenes. For these reasons, efficient methods are needed for targeting andcontrol of insertion of nucleotide sequences to be integrated into aplant genome.

SUMMARY OF THE INVENTION

[0008] Compositions and methods for the targeted integration ofnucleotide sequences into a transformed plant are provided. Thecompositions comprise transfer cassettes which are flanked bynon-identical recombination sites.

[0009] The methods find use in targeting the integration of nucleotidesequences of interest to a specific chromosomal site, finding optimalintegration sites in a plant genome, comparing promoter activity intransformed plants, engineering chromosomal rearrangements, and othergenetic manipulation of plants.

[0010] Novel minimal recombination sites (FRT) are provided for use inthe methods of the invention. Also provided are targeting cassettes andtransgenic plants and plant cells containing corresponding non-identicalrecombination sites.

BRIEF DESCRIPTION OF THE FIGURES

[0011]FIG. 1 provides one scheme for gene stacking via site-specificintegration using the FLP system.

[0012]FIG. 2 provides a construct of the representative plasmidPHP10616.

DETAILED DESCRIPTION OF THE INVENTION

[0013] Compositions and methods for the directional, targetedintegration of exogenous nucleotides into a transformed plant areprovided. The methods use novel recombination sites in a gene targetingsystem which facilitates directional targeting of desired genes andnucleotide sequences into corresponding recombination sites previouslyintroduced into the target plant genome.

[0014] In the methods of the invention, a nucleotide sequence flanked bytwo non-identical recombination sites is introduced into the targetorganism's genome establishing a target site for insertion of nucleotidesequences of interest. Once a stable plant or cultured tissue isestablished a second construct, or nucleotide sequence of interest,flanked by corresponding recombination sites as those flanking thetarget site, is introduced into the stably transformed plant or tissuesin the presence of a recombinase protein. This process results inexchange of the nucleotide sequences between the non-identicalrecombination sites of the target site and the transfer cassette.

[0015] It is recognized that the transformed plant may comprise multipletarget sites; i.e., sets of non-identical recombination sites. In thismanner, multiple manipulations of the target site in the transformedplant are available. By target site in the transformed plant is intendeda DNA sequence that has been inserted into the transformed plant'sgenome and comprises non-identical recombination sites.

[0016] Examples of recombination sites for use in the invention areknown in the art and include FRT sites (See, for example, Schlake andBode (1994) Biochemistry 33:12746-12751; Huang et al. (1991) NucleicAcids Research 19:443-448; Sadowski, Paul D. (1995) In Progress inNucleic Acid Research and Molecular Biology vol. 51, pp. 53-91; MichaelM. Cox (1989) In Mobile DNA, Berg and Howe (eds) American Society ofMicrobiology, Washington D.C., pp. 116-670; Dixon et al. (1995)18:449-458; Umlauf and Cox (1988) The EMBO Journal 7:1845-1852; Buchholzet al. (1996) Nucleic Acids Research 24:3118-3119; Kilby et al. (1993)Trends Genet. 9:413-421: Rossant and Geagy (1995) Nat. Med. 1: 592-594;Albert et al. (1995) The Plant J. 7:649-659: Bayley et al. (1992) PlantMol. Biol. 18:353-361; Odell et al. (1990) Mol. Gen. Genet. 223:369-378;and Dale and Ow (1991) Proc. Natl. Acad. Sci. USA 88:10558-105620; allof which are herein incorporated by reference.); Lox (Albert et al.(1995) Plant J. 7:649-659; Qui et al. (1994) Proc. Natl. Acad. Sci. USA91:1706-1710; Stuurman et al. (1996) Plant Mol. Biol. 32:901-913; Odellet al. (1990) Mol. Gen. Gevet. 223:369-378; Dale et al. (1990) Gene91:79-85; and Bayley et al. (1992) Plant Mol. Biol. 18:353-361.)

[0017] The two-micron plasmid found in most naturally occurring strainsof Saccharomyces cerevisiae, encodes a site-specific recombinase thatpromotes an inversion of the DNA between two inverted repeats. Thisinversion plays a central role in plasmid copy-number amplification. Theprotein, designated FLP protein, catalyzes site-specific recombinationevents. The minimal recombination site (FRT, SEQ ID NO:1) has beendefined and contains two inverted 13-base pair (bp) repeats surroundingan asymmetric 8-bp spacer. The FLP protein cleaves the site at thejunctions of the repeats and the spacer and is covalently linked to theDNA via a 3′ phosphate.

[0018] Site specific recombinases like FLP cleave and religate DNA atspecific target sequences, resulting in a precisely definedrecombination between two identical sites. To function, the system needsthe recombination sites and the recombinase. No auxiliary factors areneeded. Thus, the entire system can be inserted into and function inplant cells.

[0019] The yeast FLP\FRT site specific recombination system has beenshown to function in plants. To date, the system has been utilized forexcision of unwanted DNA. See, Lyznik et al. (1993) Nucleic Acid Res.21:969-975. In contrast, the present invention utilizes non-identicalFRTs for the exchange, targeting, arrangement, insertion and control ofexpression of nucleotide sequences in the plant genome.

[0020] To practice the methods of the invention, a transformed organismof interest, particularly a plant, containing a target site integratedinto its genome is needed. The target site is characterized by beingflanked by non-identical recombination sites. A targeting cassette isadditionally required containing a nucleotide sequence flanked bycorresponding non-identical recombination sites as those sites containedin the target site of the transformed organism. A recombinase whichrecognizes the non-identical recombination sites and catalyzessite-specific recombination is required.

[0021] It is recognized that the recombinase can be provided by anymeans known in the art. That is, it can be provided in the organism orplant cell by transforming the organism with an expression cassettecapable of expressing the recombinase in the organism, by transientexpression; or by providing messenger RNA (mRNA) for the recombinase orthe recombinase protein.

[0022] By “non-identical recombination sites” is intended that theflanking recombination sites are not identical in sequence and will notrecombine or recombination between the sites will be minimal. That is,one flanking recombination site may be a FRT site where the secondrecombination site may be a mutated FRT site. The non-identicalrecombination sites used in the methods of the invention prevent orgreatly suppress recombination between the two flanking recombinationsites and excision of the nucleotide sequence contained therein.Accordingly, it is recognized that any suitable non-identicalrecombination sites may be utilized in the invention, including FRT andmutant FRT sites, FRT and LOX sites, LOX and mutant LOX sites, as wellas other recombination sites known in the art.

[0023] By suitable non-identical recombination site implies that in thepresence of active recombinase, excision of sequences between twonon-identical recombination sites occurs, if at all, with an efficiencyconsiderably lower than the recombinationally-mediated exchangetargeting arrangement of nucleotide sequences into the plant genome.Thus, suitable non-identical sites for use in the invention includethose sites where the efficiency of recombination between the sites islow; for example, where the efficiency is less than about 30 to about50%, preferably less than about 10 to about 30%, more preferably lessthan about 5 to about 10%.

[0024] As noted above, the recombination sites in the targeting cassettecorrespond to those in the target site of the transformed plant. Thatis, if the target site of the transformed plant contains flankingnon-identical recombination sites of FRT and a mutant FRT, the targetingcassette will contain the same FRT and mutant FRT non-identicalrecombination sites.

[0025] It is furthermore recognized that the recombinase, which is usedin the invention, will depend upon the recombination sites in the targetsite of the transformed plant and the targeting cassette. That is, ifFRT sites are utilized, the FLP recombinase will be needed. In the samemanner, where lox sites are utilized, the Cre recombinase is required.If the non-identical recombination sites comprise both a FRT and a loxsite, both the FLP and Cre recombinase will be required in the plantcell.

[0026] The FLP recombinase is a protein that catalyzes a site-specificreaction that is involved in amplifying the copy number of the twomicron plasmid of S. cerevisiae during DNA replication. FLP protein hasbeen cloned and expressed. See, for example, Cox (1993) Proc. Natl.Acad. Sci. U.S.A. 80:4223-4227. The FLP recombinase for use in theinvention may be that derived from the genus Saccharomyces. It may bepreferable to synthesize the recombinase using plant preferred codonsfor optimum expression in a plant of interest. See, for example, U.S.application Ser. No. 08/972,258 filed Nov. 18, 1997, entitled “NovelNucleic Acid Sequence Encoding FLP Recombinase” now U.S. Pat. No.5,929,301, herein incorporated by reference.

[0027] The bacteriophage recombinase Cre catalyzes site-specificrecombination between two lox sites. The Cre recombinase is known in theart. See, for example, Guo et al. (1997) Nature 389:40-46; Abremski etal. (1984) J. Biol. Chem. 259:1509-1514; Chen et al. (1996) Somat. CellMol. Genet. 22:477-488; and Shaikh et al. (1977) J. Biol. Chem.272:5695-5702. All of which are herein incorporated by reference. SuchCre sequence may also be synthesized using plant preferred codons.

[0028] Where appropriate, the nucleotide sequences to be inserted in theplant genome may be optimized for increased expression in thetransformed plant. Where mammalian, yeast, or bacterial genes are usedin the invention, they can be synthesized using plant preferred codonsfor improved expression. It is recognized that for expression inmonocots, dicot genes can also be synthesized using monocot preferredcodons. Methods are available in the art for synthesizing plantpreferred genes. See, for example, U.S. Pat. Nos. 5,380,831, 5,436,391,and Murray et al. (1989) Nucleic Acids Res. 17:477-498, hereinincorporated by reference.

[0029] The plant preferred codons may be determined from the codonsutilized more frequently in the proteins expressed in the plant ofinterest. It is recognized that monocot or dicot preferred sequences maybe constructed as well as plant preferred sequences for particular plantspecies. See, for example, EPA 0359472; EPA 0385962; WO 91/16432; Perlaket al. (1991) Proc. Natl. Acad. Sci. USA, 88:3324-3328; and Murray etal. (1989) Nucleic Acids Research, 17: 477-498. U.S. Pat. No. 5,380,831;U.S. Pat. No. 5,436,391; and the like, herein incorporated by reference.It is further recognized that all or any part of the gene sequence maybe optimized or synthetic. That is, fully optimized or partiallyoptimized sequences may also be used.

[0030] Additional sequence modifications are known to enhance geneexpression in a cellular host and can be used in the invention. Theseinclude elimination of sequences encoding spurious polyadenylationsignals, exon-intron splice site signals, transposon-like repeats, andother such well-characterized sequences, which may be deleterious togene expression. The G-C content of the sequence may be adjusted tolevels average for a given cellular host, as calculated by reference toknown genes expressed in the host cell. When possible, the sequence ismodified to avoid predicted hairpin secondary mRNA structures.

[0031] The present invention also encompasses novel FLP recombinationtarget sites (FRT). The FRT (SEQ ID NO:1) has been identified as aminimal sequence comprising two 13 base pair repeats, separated by an 8base spacer, as follows:

[0032] 5′-GAAGTTCCTATTC[TCTAGAAA]GTATAGGAACTTC3′

[0033] wherein the nucleotides within the brackets indicate the spacerregion. The nucleotides in the spacer region can be replaced with acombination of nucleotides, so long as the two 13-base repeats areseparated by eight nucleotides. It appears that the actual nucleotidesequence of the spacer is not critical, however for the practice of theinvention, some substitutions of nucleotides in the space region maywork better than others.

[0034] The eight base pair spacer is involved in DNA-DNA pairing duringstrand exchange. The asymmetry of the region determines the direction ofsite alignment in the recombination event, which will subsequently leadto either inversion or excision. As indicated above, most of the spacercan be mutated without a loss of function. See, for example, Schlake andBode (1994) Biochemistry 33:12746-12751, herein incorporated byreference.

[0035] Novel FRT mutant sites are provided for use in the practice ofthe methods of the present invention. Such mutant sites may beconstructed by PCR-based mutagenesis. While mutant FRT sites (SEQ IDNOS:2, 3, 4 and 5) are provided herein, it is recognized that othermutant FRT sites may be used in the practice of the invention. Thepresent invention is not the use of a particular FRT or recombinationsite, but rather that non-identical recombination sites or FRT sites canbe utilized for targeted insertion and expression of nucleotidesequences in a plant genome. Thus, other mutant FRT sites can beconstructed and utilized based upon the present disclosure.

[0036] As discussed above, bringing genomic DNA containing a target sitewith non-identical recombination sites together with a vector containinga transfer cassette with corresponding non-identical recombinationsites, in the presence of the recombinase, results in recombination. Thenucleotide sequence of the transfer cassette located between theflanking recombination sites is exchanged with the nucleotide sequenceof the target site located between the flanking recombination sites. Inthis manner, nucleotide sequences of interest may be preciselyincorporated into the genome of the host.

[0037] It is recognized that many variations of the invention can bepracticed. For example, target sites can be constructed having multiplenon-identical recombination sites. Thus, multiple genes or nucleotidesequences can be stacked or ordered at precise locations in the plantgenome. Likewise, once a target site has been established within thegenome, additional recombination sites may be introduced byincorporating such sites within the nucleotide sequence of the transfercassette and the transfer of the sites to the target sequence. Thus,once a target site has been established, it is possible to subsequentlyadd sites, or alter sites through recombination.

[0038] Another variation includes providing a promoter or transcriptioninitiation region operably linked with the target site in an organism.Preferably, the promoter will be 5′ to the first recombination site. Bytransforming the organism with a transfer cassette comprising a codingregion, expression of the coding region will occur upon integration ofthe transfer cassette into the target site. This embodiment provides fora method to select transformed cells, particularly plant cells, byproviding a selectable marker sequence as the coding sequence.

[0039] Other advantages of the present system include the ability toreduce the complexity of integration of trans-genes or transferred DNAin an organism by utilizing transfer cassettes as discussed above andselecting organisms with simple integration patterns. In the samemanner, preferred sites within the genome can be identified by comparingseveral transformation events. A preferred site within the genomeincludes one that does not disrupt expression of essential sequences andprovides for adequate expression of the transgene sequence.

[0040] The methods of the invention also provide for means to combinemultiple cassettes at one location within the genome. See, for example,FIG. 1. Recombination sites may be added or deleted at target siteswithin the genome.

[0041] Any means known in the art for bringing the three components ofthe system together may be used in the invention. For example, a plantcan be stably transformed to harbor the target site in its genome. Therecombinase may be transiently expressed or provided. Alternatively, anucleotide sequence capable of expressing the recombinase may be stablyintegrated into the genome of the plant. In the presence of thecorresponding target site and the recombinase, the transfer cassette,flanked by corresponding non-identical recombination sites, is insertedinto the transformed plant's genome.

[0042] Alternatively, the components of the system may be broughttogether by sexually crossing transformed plants. In this embodiment, atransformed plant, parent one, containing a target site integrated inits genome can be sexually crossed with a second plant, parent two, thathas been genetically transformed with a transfer cassette containingflanking non-identical recombination sites, which correspond to those inplant one. Either plant one or plant two contains within its genome anucleotide sequence expressing recombinase. The recombinase may be underthe control of a constitutive or inducible promoter.

[0043] Inducible promoters include heat-inducible promoters,estradiol-responsive promoters, chemical inducible promoters, and thelike. Pathogen inducible promoters include those frompathogenesis-related proteins (PR proteins), which are induced followinginfection by a pathogen; e.g., PR proteins, SAR proteins,beta-1,3-glucanase, chitinase, etc. See, for example, Redolfi et al.(1983) Neth. J. Plant Pathol. 89:245-254; Uknes et al. (1992) The PlantCell 4:645-656; and Van Loon (1985) Plant Mol. Virol. 4:111-116. In thismanner, expression of recombinase and subsequent activity at therecombination sites can be controlled.

[0044] Constitutive promoters for use in expression of genes in plantsare known in the art. Such promoters include, but are not limited to 35Spromoter of cauliflower mosaic virus (Depicker et al. (1982) Mol. Appl.Genet. 1:561-573; Odell et al. (1985) Nature 313:810-812), ubiquitinpromoter (Christensen et al. (1992) Plant Mol. Biol. 18:675-689),promoters from genes such as ribulose bisphosphate carboxylase (DeAlmeida et al. (1989) Mol. Gen. Genet. 218:78-98), actin (McElroy et al.(1990) Plant J. 2:163-171), histone, DnaJ (Baszczynski et al. (1997)Maydica 42:189-201), and the like.

[0045] The compositions and methods of the invention find use intargeting the integration of transferred nucleotide sequences to aspecific chromosomal site. The nucleotide sequence may encode anynucleotide sequence of interest. Particular genes of interest includethose which provide a readily analyzable functional feature to the hostcell and/or organism, such as marker genes, as well as other genes thatalter the phenotype of the recipient cells, and the like. Thus, geneseffecting plant growth, height, susceptibility to disease, insects,nutritional value, and the like may be utilized in the invention. Thenucleotide sequence also may encode an “antisense” sequence to turn offor modify gene expression.

[0046] It is recognized that the nucleotide sequences will be utilizedin a functional expression unit or cassette. By functional expressionunit or cassette is intended, the nucleotide sequence of interest with afunctional promoter, and in most instances a termination region. Thereare various ways to achieve the functional expression unit within thepractice of the invention. In one embodiment of the invention, thenucleic acid of interest is transferred or inserted into the genome as afunctional expression unit. Alternatively, the nucleotide sequence maybe inserted into a site within the genome which is 3′ to a promoterregion. In this latter instance, the insertion of the coding sequence 3′to the promoter region is such that a functional expression unit isachieved upon integration.

[0047] For convenience, for expression in plants, the nucleic acidencoding target sites and the transfer cassettes, including thenucleotide sequences of interest, can be contained within expressioncassettes. The expression cassette will comprise a transcriptionalinitiation region, or promoter, operably linked to the nucleic acidencoding the peptide of interest. Such an expression cassette isprovided with a plurality of restriction sites for insertion of the geneor genes of interest to be under the transcriptional regulation of theregulatory regions.

[0048] The transcriptional initiation region, the promoter, may benative or homologous or foreign or heterologous to the host, or could bethe natural sequence or a synthetic sequence. By foreign is intendedthat the transcriptional initiation region is not found in the wild-typehost into which the transcriptional initiation region is introduced.Either a native or heterologous promoter may be used with respect to thecoding sequence of interest.

[0049] The transcriptional cassette will include in the 5′-3′ directionof transcription, a transcriptional and translational initiation region,a DNA sequence of interest, and a transcriptional and translationaltermination region functional in plants. The termination region may benative with the transcriptional initiation region, may be native withthe DNA sequence of interest, or may be derived from another source.Convenient termination regions are available from the potato proteinaseinhibitor (PinII) gene or from Ti-plasmid of A. tumefaciens, such as theoctopine synthase and nopaline synthase termination regions. See also,Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991)Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen etal. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158;Ballas et al. 1989) Nucleic Acids Res. 17:7891-7903; Joshi et al. (1987)Nucleic Acid Res. 15:9627-9639.

[0050] The expression cassettes may additionally contain 5′ leadersequences in the expression cassette construct. Such leader sequencescan act to enhance translation. Translation leaders are known in the artand include: picomavirus leaders, for example, EMCV leader(Encephalomyocarditis 5′ noncoding region) (Elroy-Stein, et al. (1989)PNAS USA, 86:6126-6130); potyvirus leaders, for example, TEV leader(Tobacco Etch Virus) (Allison et al. (1986); MDMV leader (Maize DwarfMosaic Virus); Virology, 154:9-20), and human immunoglobulin heavy-chainbinding protein (BiP), (Macejak and Sarnow (1991) Nature, 353:90-94;untranslated leader from the coat protein mRNA of alfalfa mosaic virus(AMV RNA 4), (Jobling and Gehrke (1987) Nature, 325:622-625; tobaccomosaic virus leader (TMV), (Gallie et al. (1989) Molecular Biology ofRNA, pages 237-256, Gallie et al. (1987) Nucl. Acids Res. 15:3257-3273;and maize chlorotic mottle virus leader (MCMV) (Lommel, S. A. et al.(1991) Virology, 81:382-385). See also, Della-Cioppa et al. (1987) PlantPhysiology, 84:965-968. Other methods known to enhance translation canalso be utilized, for example, introns, and the like.

[0051] The expression cassettes may contain one or more than one gene ornucleic acid sequence to be transferred and expressed in the transformedplant. Thus, each nucleic acid sequence will be operably linked to 5′and 3′ regulatory sequences. Alternatively, multiple expressioncassettes may be provided.

[0052] Generally, the expression cassette will comprise a selectablemarker gene for the selection of transformed cells. Selectable markergenes are utilized for the selection of transformed cells or tissues.

[0053] See generally, Yarranton, G. T. (1992) Curr. Opin. Biotech.,3:506-511; Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA,89:6314-6318; Yao et al. (1992) Cell, 71:63-72; Reznikoff, W. S. (1992)Mol. Microbiol., 6:2419-2422; Barkley et al. (1980) The Operon, pp.177-220; Hu et al. (1987) Cell, 48:555-566; Brown et al. (1987) Cell,49:603-612; Figge et al (1988) Cell, 52:713-722; Deuschle et al. (1989)Proc. Natl. Acad. Aci. USA, 86:5400-5404; Fuerst et al. (1989) Proc.Natl. Acad. Sci. USA, 86:2549-2553; Deuschle et al (1990) Science,248:480-483; Gossen, M. (1993) PhD Thesis, University of Heidelberg;Reines et al. (1993) Proc. Natl. Acad. Sci. USA, 90:1917-1921; Labow etal. (1990) Mol. Cell Bio., 10:3343-3356; Zambretti et al. (1992) Proc.Natl. Acad. Sci. USA, 89:3952-3956; Baim et al. (1991) Proc. Natl. Acad.Sci. USA, 88:5072-5076; Wyborski et al. (1991) Nuc. Acids Res.,19:4647-4653; Hillenand-Wissman, A. (1989) Topics in Mol. and Struc.Biol., 10:143-162; Degenkolb et al. (1991) Antimicrob. AgentsChemother., 35:1591-1595; Kleinschnidt et al. (1988) Biochemistry,27:1094-1104; Gatz et al. (1992) Plant J., 2:397-404; Bonin, A. L.(1993) PhD Thesis, University of Heidelberg; Gossen et al. (1992) Proc.Natl. Acad. Sci. USA, 89:5547-5551; Oliva et al. (1992) Antimicrob.Agents Chemother., 36:913-919; Hlavka et al. (1985) Handbook of Exp.Pharmacology, 78; Gill et al. (1988) Nature 334:721-724. Suchdisclosures are herein incorporated by reference.

[0054] The methods of the invention can also be utilized to find optimalintegration sites within a plant genome. In this manner, a plant istransformed with an expression cassette comprising a selectable markergene. The expression cassette is a target site as the marker gene isflanked by non-identical recombination sites. Transformed protoplast,tissues, or whole plants can be tested to determine the levels ofactivity of the inserted gene. By comparison of cellular activities ofthe gene in different insertion sites, preferred integration sites maybe found wherein the gene is expressed at high or acceptable levels.These plants can then be utilized with subsequent retargeting techniquesto replace the marker gene with other genes or nucleotide sequences ofinterest. In the same manner, multiple genes may be inserted at theoptimal site for expression. See, for example, FIG. 2 which sets forthone scheme for gene stacking utilizing site-specific integration usingthe FRT/FLP system.

[0055] A variety of genetic manipulations are available using thecompositions of the present invention including, for example, comparingpromoter activity in a transformed plant. Prior to the presentinvention, promoter activity could not accurately be assessed andcompared because the chimeric genes were inserted at different locationswithin the plant genome. Such chromosomal locations affected activity.By utilizing the methods of the present invention, a direct comparisonof promotor activity in a defined chromosomal context is possible. Thus,using the methods, enhanced activity of genes can be achieved byselecting optimal chromosomal sites as well as optimal promoters forexpression in the plant cell.

[0056] The present invention may be used for transformation of any plantspecies, including but not limited to corn (Zea mays), canola (Brassicanapus, Brassica rapa ssp.), alfalfa (Medicago sativa), rice (Oryzasativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghumvulgare), sunflower (Helianthus annuus), wheat (Triticum aestivum),soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum),sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee(Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidentale),macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugarbeets (Beta vulgaris), oats, barley, vegetables, ornamentals, andconifers.

[0057] Vegetables include tomatoes (Lycopersicon esculentum), lettuce(e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans(Phaseolus limensis), peas (Lathyrus spp.) and members of the genusCucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis),and musk melon (C. melo). Ornamentals include azalea (Rhododendronspp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscusrosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils(Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthuscaryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum.Conifers which may be employed in practicing the present inventioninclude, for example, pines such as loblolly pine (Pinus taeda), slashpine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine(Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir(Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitkaspruce (Picea glauca); redwood (Sequoia sempervirens); true firs such assilver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedarssuch as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis). Preferably, plants of the presentinvention are crop plants (for example, corn, alfalfa, sunflower,canola, soybean, cotton, peanut, sorghum, wheat, tobacco, etc.), morepreferably corn and soybean plants, yet more preferably corn plants.

[0058] It is recognized that the methods of the invention may be appliedin any plant system. Methods for transformation of plants are known inthe art. In this manner, genetically modified plants, plant cells, planttissue, seed, and the like can be obtained. Transformation protocols mayvary depending on the type of plant or plant cell, i.e., monocot ordicot, targeted for transformation. Suitable methods of transformingplant cells include microinjection (Crossway et al. (1986) Biotechniques4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci.USA, 83:5602-5606, Agrobacterium mediated transformation (Hinchee et al.(1988) Biotechnology, 6:915-921), direct gene transfer (Paszkowski etal. (1984) EMBO J., 3:2717-2722), and ballistic particle acceleration(see, for example, Sanford et al., U.S. Pat. No. 4,945,050; WO91/10725and McCabe et al. (1988) Biotechnology, 6:923-926). Also see, Weissingeret al. (1988) Annual Rev. Genet., 22:421-477; Sanford et al. (1987)Particulate Science and Technology, 5:27-37 (onion); Christou et al.(1988) Plant Physiol. 87:671-674(soybean); McCabe et al. (1988)Bio/Technology, 6:923-926 (soybean); Datta et al. (1990) Biotechnology,8:736-740(rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA,85:4305-4309(maize); Klein et al. (1988) Biotechnology, 6:559-563(maize); WO91/10725 (maize); Klein et al. (1988) Plant Physiol.,91:440-444(maize); Fromm et al. (1990) Biotechnology, 8:833-839; andGordon-Kamm et al. (1990) Plant Cell, 2:603-618 (maize); Hooydaas-VanSlogteren & Hooykaas (1984) Nature (London), 311:763-764; Bytebier etal. (1987) Proc. Natl. Acad. Sci. USA, 84:5345-5349 (Liliaceae); De Wetet al. (1985) In The Experimental Manipulation of Ovule Tissues, ed. G.P. Chapman et al., pp. 197-209. Longman, N Y (pollen); Kaeppler et al.(1990) Plant Cell Reports, 9:415-418; and Kaeppler et al. (1992) Theor.Appl. Genet., 84:560-566 (whisker-mediated transformation); D'Halluin etal. (1992) Plant Cell, 4:1495-1505 (electroporation); Li et al. (1993)Plant Cell Reports, 12:250-255 and Christou and Ford (1995) Annals ofBotany, 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology,14:745-750 (maize via Agrobacterium tumefaciens); all of which areherein incorporated by reference.

[0059] The cells which have been transformed may be grown into plants inaccordance with conventional approaches. See, for example, McCormick etal. (1986) Plant Cell Reports, 5:81-84. These regenerated plants maythen be pollinated with either the same transformed strain or differentstrains, and the resulting hybrid having the desired phenotypiccharacteristic identified. Two or more generations may be grown toensure that the subject phenotypic characteristic is stably maintainedand inherited and then seeds harvested to ensure the desired phenotypeor other property has been achieved.

[0060] It is recognized that any means of transformation may be utilizedfor the present invention. However, for inserting the target site withinthe transformed plant, Agrobacterium-mediated transformation may bepreferred. Agrobacterium-mediated transformation generally tends toinsert a lower copy number of transferred DNA than does particlebombardment or other transformation means.

[0061] The following examples are offered by way of illustration and notby way of limitation.

EXPERIMENTAL

[0062] The general present invention provides a procedure for usingexisting and novel FRT sites in a new gene targeting system whichfacilitates directional retargeting of desired genes into FRT sitespreviously introduced in the target organism's genome. The novel FRTsites differ from previously described FRT sites in the sequence of the8 bp spacer regions of the FRT sites. Previous publications also haveshown that in the presence of FLP protein, recombination of sequencesbetween two FRT sites occurs efficiently only with two identical FRTsites. See for example Umlauf and Cox (1988) Embo J. 7:1845-1852;Schlake and Bode (1994) Biochem. 33:12746-12751. To use the invention, agene or DNA sequence is flanked by two non-identical FRT sites andintroduced into a target organism's genome. The enclosed gene can be aselectable marker, thereby allowing selection for successfullyintroduced sequences. Molecular characterization confirms integration ofdesired sequences including complete FRT sites. Listed below are genericexamples of vector constructions useful in practicing the invention:

[0063] A. FRTa-P1-G1-T1-FRTb

[0064] B. FRTa-P1-G1-T1-FRTa

[0065] C. FRTb-P1-G1-T1-FRTb

[0066] D. P1-FRTa-G1-T1-FRTb

[0067] E. P1-FRTa-G1-T1-FRTa

[0068] F. P1-FRTb-G1-T1-FRTb

[0069] G. P1-ATG::FRTa::G1(noATG)-T1-P2-G2-T2-FRTb

[0070] H. P1-ATG::FRTa::G1(noATG)-T1-P2-G2-T2-FRTb-P3-G3-T3

[0071] I. P1-ATG::FRTa::G1(noATG)-T1-FRTa::G2(noATG)-T2-FRTb

[0072] J. P1-ATG::FRTa::G1(noATG)-T1-FRTa::G2(noATG)-T2-FRTb-P3-G3-T3

[0073] K. P1-FRTa-G1-T1-P2-G2-T2-FRTb

[0074] L. P1-FRTa-G1-T1-P2-G2-T2-FRTb-P3-G3-T3

[0075] M. P1-FRTa-G1-T1-FRTa-G2-T2-FRTb

[0076] N. P1-FRTa-G1-T1-FRTa-G2-T2-FRTb-P3-G3-T3

[0077] Variations thereof may be constructed with other promoters,genes, terminators or FRT sites.

[0078] FRTa and FRTb are two examples of non-identical FRT sites. P1, P2and P3 are different promoters, G1, G2, and G3 are different genes, T1,T2 and T3 are different terminators. ATG is the start of translationcodon for the subsequent gene. The designation noATG indicates thatparticular gene is devoid of the ATG translation start codon. The symbol:: implies a fusion between adjacent elements, and where used betweenATG, FRT and a gene, implies that the sequences are put together togenerate an in frame translation fusion that results in a properlyexpressed and functional gene product.

[0079] A to F are preferred configurations for testing new FRT sites forability to recombine sequences between them; the desired situation beingthat when two of the same site are used, recombination is efficient andthat when two different sites are used, no recombination between themtakes place in the presence of FLP protein. G to J are preferredconfigurations for general use in developing lines for retargeting. Itis understood that any number of genes or other combinations ofsequences can be assembled for use as part of this invention. K to N arepossible configurations that could be used also.

[0080] Once a stable plant or cultured tissue is established with one ofthe constructs above, a second construct flanked by the same FRT sitesused to flank the sequences in the first construct above is introducedinto the stably transformed tissues in conjunction with the expressionof FLP protein. The new vector constructs can be, but are not limited tothe following:

[0081] O. FRTa::G1(noATG)-T1-FRTb

[0082] P. FRTa::G1(noATG)-T1-P2-G2-T2-FRTb

[0083] Q. FRTa-G1-T1-FRTb

[0084] R. FRTa-G1-T1-P2-G2-T2-FRTb

[0085] The FLP protein can be supplied by a) co-transforming with aplasmid carrying a gene encoding FLP; b) co-introducing FLP mRNA orprotein directly; c) using a line for the initial transformation thatexpresses FLP either constitutively or following induction; or d)growing out the plants carrying the initial targeted vectors, crossingto plants that express active FLP protein and selecting events in theprogeny.

[0086] As a working example, sequence O above is introduced into a linecontaining a copy of sequence G stably integrated in the genome, in thepresence of functional FLP protein. Recombination takes place betweenidentical FRT sites such that the sequence between FRT sites in Oreplaces the sequence between the corresponding FRT sites of sequence G,thereby yielding a directionally targeted reintegrated new sequence. Thenew gene in O is now driven off of the P1 promoter in G. The purpose fordesigning some of the constructs without an ATG start codon on the geneis so that if random integration occurs, there is an extremely lowprobability of expression of the introduced gene, since in order forthis to happen, the fragment would need to integrate behind anendogenous promoter region and in the correct reading frame. This wouldoccur extremely rarely and our data to date have yielded no examples ofthis happening using a sequence such as O where the contained gene isthe easily scorable GUS gene. One requirement for each gene to beconstructed in this way (i.e., no ATG on the gene but with the ATGupstream of the FRT site) is the demonstration that the gene cantolerate a fusion of the FRT sequence between the ATG codon and thesecond codon of the protein. To date this has worked for quite a numberbut not all genes; in the latter cases the other form of the constructretaining the ATG (for example Q) could be used. All of the sequenceslisted above are expected to work in this scheme, some at differentfrequencies or efficiencies than others.

[0087] One problem this strategy addresses is limitations with currenttransformation approaches, particularly in plants, where delivery of DNAinto cells or nuclei and subsequent integration in the genome occursmore or less randomly and unpredictably. This is particularly true withparticle bombardment methods; arguments have been made thatAgrobacterium-based methods tend to deliver T-DNA border-flankedsequences to more actively transcribed regions of the genome, but beyondthat the process is still largely random. Therefore, for commercialproduct development, large numbers (estimates of >200) of events need tobe generated in order to identify one event: a) that expresses at thedesired level; b) where the gene product is functional and efficacious;c) which has a simple integration complexity to facilitate breeding; d)which does not contain extraneous sequences posing possible regulatoryconcerns; e) which maintains stability in expression over generations;f) most importantly, which does not have a negative impact on agronomicperformance characteristics when carried through a breeding programinvolving introgression of the trait into different genetic backgrounds.Resource utilization is very large and so schemes that can markedlyreduce the resource demand would be very beneficial to production oflarger numbers of desired final products.

EXAMPLE 1 Creation of Novel Non-Identical FRT Sites

[0088] DNA fragments containing novel FRT sequences were constructedeither by synthesizing, annealing and ligating complementaryoligonucleotides or by creating primers for PCR amplification (Mullisand Faloona, 1987) of a DNA product containing the new FRT sequence nearthe 5′ end of the PCR product. The newly constructed FRT productincludes flanking restriction sites useful for cloning into plantexpression units. In general, the 5′ end is flanked by an NheI site anda terminal NcoI site. The NcoI site includes the bases ATG, which areadvantageously used in newly developed vector constructs as therecognition sequence to initiate an open reading frame. Insequence-based constructs designated noATG/IFRT, the NheI site is usedfor cloning thereby eliminating the upstream ATG in the process. At the3′ end of the FRT sequence, a restriction site is included enablingunique identification of the individual spacer sequences. As specificexamples, the wild type FRT site (designated FRT1 here) is cloned with aflanking BglII site, the FRT5 site (spacer TTCAAAAG) (nt 39-46 of SEQ IDNO:3) has a ScaI site, the FRT6 site (spacer TTCAAAAA) (nt 36-49 of SEQID NO:4) has an AatII site, and the FRT7 site (spacer TTCAATAA) (nt36-46 of SEQ ID NO:5) has an SpeI site. The outermost flankingrestriction site is an XhoI site and is used to clone a gene of interestinto the open reading frame.

[0089] The structures and sequences of the FRT sites as designed and/orused in the present invention example are depicted below with positionsof restriction sites, repeats and spacer regions indicated. FRT1NcoI   NheI   Repeat 1           Repeat 2         Spacer     InvertedRepeat    BglII XhoI (SEQ ID NO:2) 5′        CCATGGCTAGC GAAGTTCCTATTCCGAAGTTCCTATTC TCTAGAAA GTATAGGAACTTC AGATCTCGAG FRT5NcoI   NheI   Repeat 1           Repeat 2         Spacer     InvertedRepeat    ScaI XhoI (SEQ ID NO:3) 5′        CCATGGCTAGC GAAGTTCCTATTTCCGAAGTTCCTATTC TTCAAAAG GTATAGGAACTTC AGTACTCGAG FRT6 NcoI   NheI   Repeat 1           Repeat 2         Spacer     InvertedRepeat     AatII   XhoI (SEQ ID NO:4) 5′       CCATGGCTAGCGAAGTTCCTATTCC GAAGTTCCTATTC TTCAAAAA GTATAGGAACTTC AGACGTCCTCGAG FRT7NcoI   NheI   Repeat 1           Repeat 2         Spacer    InvertedRepeat   SpeI    XhoI (SEQ ID NO:5) 5′        CCATGGCTAGC GAAGTTCCTATTCCGAAGTTCCTATTCTTCAATAA GTATAGGAACTTCACTAGTTCTCGAG

EXAMPLE 2 Creation of Plant Transformation Vectors Containing NovelNon-Identical FRT Sites

[0090] Based on the design of FRT sites as described above, PCR orstandard mutagenesis protocols were used to create an XhoI siteoverlapping the start of a gene sequence to be used for cloningdownstream of the FRT site, thereby converting the ATG start codon toGTG. Ligation of an FRT to the mutated gene sequence at XhoI creates anew open reading frame initiating 5′ to the FRT. A second FRT sequencecan be cloned downstream of the terminator using a variety of methodsincluding PCR or ligation. The FRT/gene/terminator/FRT unit can then beused to make target or substrate constructs.

[0091] Targets are created by inserting a promoter at the NcoI siteupstream of the first FRT. This maintains a complete open reading frameof the FRT/gene fusion. These target constructs are for use intransformation experiments to create desirable “target lines”. Substratevectors are constructed by cloning with the NheI site to truncate thestart codon of the FRT/gene unit, thereby eliminating the proper openreading frame. These substrate vectors are used in experiments designedto retarget a new gene flanked by FRT sites into the corresponding FRTsites previously introduced in the target lines. In either case, tocreate multiple gene cassettes, additional promoter/gene/terminatorunits are inserted between the terminator and the second FRT in eithertarget or substrate molecules.

EXAMPLE 3 Demonstration of Functionality of Novel FRT Sites andRequirement for Two Identical Sites for Efficient Recombination of DNASequences Positioned between Two FRT Sites

[0092] Plasmids containing two identical or two different FRT sequenceswere assayed for efficiency of recombination of sequences between theFRT sites by transformation into 294-FLP, a version of the E. colistrain MM294 with FLP recombinase integrated into the lacZ locus(Buchholz et al. 1996). Strains were grown overnight at 37° C. withshaking, allowing for constitutive expression of FLP recombinase in thecultures. The plasmid DNA was isolated using standard procedures anddigested with restriction enzymes that create novel restrictionfragments following FLP mediated recombination. The extent ofrecombination between FRT sites was estimated by examining bandingpatterns on an agarose gel. Table 1 summarizes data from the gelanalysis. TABLE 1 Target Site Combination Extent of Recombination FRT1and FRT1 Complete FRT5 and FRT5 Extensive, but partially incomplete FRT6and FRT6 Complete FRT7 and FRT7 Complete FRT1 and FRT5 No recombinationFRT1 and FRT6 No recombination FRT1 and FRT7 No recombination FRT5 andFRT6 No recombination FRT5 and FRT7 No recombination FRT6 and FRT7 Verysmall amount of recombination

[0093] The results from these studies indicate that excision ofsequences between identical FRT sites occurs with high efficiency ingeneral (FRT5, SEQ ID NO:3, appeared to be less efficient overall thanFRT1, SEQ ID NO:2, or the novel FRT6, SEQ ID NO:4, and FRT 7, SEQ IDNO:5, sites). As importantly, recombination with two different FRT siteswas absent, or at least undetectable under the conditions of this assayfor all combinations but FRT6, SEQ ID NO:4, and FRT7, SEQ ID NO:5, wherea small degree of recombination was noted. These data provided strongsupport for the potential utility of non-identical FRT sites indeveloping a directional gene integration system. A point to note isthat because recombination of sequences between two identical FRT sitescan occur with different efficiencies depending on the specific FRT siteused (e.g., FRT5, SEQ ID NO:3, in the present experiment), the design ofconstructs for directional targeted integration may require judiciousselection of pairs of FRT sites to optimize for the desiredrecombination efficiency or to avoid any unwanted recombination.

EXAMPLE 4 Introduction of DNA Sequences Which Include NovelNon-Identical FRT Sites Into Plant Cells, Generation and Recovery ofStable Transgenic Events (“Target Lines”), Preservation of “TargetLines” and Regeneration of Plants

[0094] A number of stable transgenic events carrying FRT target siteswere produced. These target lines were generated by introducing one of aseries of constructs including, for example, PHP9643, PHP10616,PHP11407, PHP11410, PHP11457, PHP11599, PHP11893 or PHP14220 (See Table2) into corn cells, either by particle bombardment, as described inRegister et al. (1994) Plant Mol. Biol. 25:951-961 or via Agrobacteriumco-cultivation as described by Heath et al. (1997) Mol. Plant-MicrobeInteract. 10:22-227; Hiei et al. (1994) Plant J. 6:271-282 and Ishida etal. (1996) Nat. Biotech. 14:745-750, and in U.S. Provisional ApplicationSerial No. 60/045,121 to “Agrobacterium Mediated SorghumTransformation”, filed Apr. 30, 1997, now U.S. application Ser. No.09/056,418, filed Apr. 7, 1998. All vectors were constructed usingstandard molecular biology techniques as described for example inSambrook et al., (1989) Molecular Cloning: A Laboratory Manual (2^(nd)ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y.). Table 2below describes the components within each of the vectors used to createa set of target lines. The assembly strategy was as follows. The firstexpression unit in each case contains the 2.0 kb PstI fragment of themaize ubiquitin promoter Ubi-1 (Christensen et al. (1992) Plant Mol.Biol. 18:675-689). Downstream of the ubiquitin promoter, varying FRTsequences were inserted using NcoI or other sites that retained the ATGstart codon. PHP10616 has the mo-PAT (U.S. Provisional PatentApplication Serial No. 60/035,560 to “Methods for ImprovingTransformation Efficiency”, filed Jan. 14, 1997 now U.S. Pat. No.6,096,947) coding sequence fused in frame at the XhoI site flanking FRT1(see above, SEQ ID NO:2). PHP11407 and PHP11893 have GFPm-C3(PCT/US97/07688 filed May 1, 1997 from Provisional Application60/016,345 filed May 1, 1996, now WO97/41228) containing the secondintron from potato ST-LS1 (Vancanneyt et al. (1990) Mol. Gen. Genet.220:245-250) fused in frame at the XhoI site of FRT1 and FRT6,respectively. The potato proteinase inhibitor II (PinII) terminator(bases 2 to 310 from An et al. (1989) Plant Cell 1:115-122) was ligateddownstream of the coding sequences. PHP10616 has an FRT5 sequence (SEQID NO:3) cloned downstream of the PinII terminator. TABLE 2 PHPUpstream-1 Coding-1 Downstream-1 Upstream-2 9643 Ubiquitin ATG/FRT1E35S/35S/O′/ADH intron 10616 Ubiquitin ATG/FRT1/moPAT pinII, FRT5 11407Ubiquitin ATG/FRT1/GFPm-C3-intron pinII Ubiquitin 11410 UbiquitinATG/FRT5 E35S/35S/O′/ADH intron 11457 Ubiquitin ATG/FRT6 E35S/35S/O′/ADHintron 11599 Ubiquitin ATG/FRT6 35S/O′/ADH intron 11893 UbiquitinATG/FRT6/GFPm-C3-intron pinII Ubiquitin 14220 Ubiquitin/FRT1 FLPm pinIIUbiquitin in 5′ UTR Ubiquitin/FRT1 FLPm pinII Ubiquitin in intron PHPCoding-2 Downstream-2 Coding-3 Downstream-3 9643 moPAT 35S termNoATG/FRT1/GFPm pinII, FRT5 10616 11407 HM1 pinII, FRT5 11410 BAR 35Sterm, FRT1 11457 BAR 35S term, FRT1 11599 BAR 35S term, FRT1 11893 HM1pinII, FRT1 14220 GFPm pinII, FRT5 GFPm pinII, FRT5

[0095] The second expression units have the maize ubiquitin promoter oralternatively either the enhanced or the standard versions of thecauliflower mosaic virus 35S promoter. The standard 35S promoterincludes bases −421 to +2 (from Gardner et al. (1981) Nucl. Acids Res.9:2871-2888), and the enhanced version has a duplication of bases −421to −90 upstream of this standard 35S promoter. The 79 bp tobacco mosaicvirus leader O' (Gallie et al. (1987) Nucl. Acids Res. 15:3257-3273) isinserted downstream of the 35S promoter followed by the first intron ofthe maize alcohol dehydrogenase ADH1-S gene (Dennis et al. (1984) Nucl.Acids Res. 12:3983-3990). Coding sequences in these second expressionunits include either mo-PAT, bar (Thompson et al. (1987) EMBO J.6:2519-2523), or HM1 (Johal and Briggs, Science 258:985-987) genesfollowed by either the PinII terminator or the 35S terminator(nucleotides 7487-7639 in Gardner et al. (1981) Nucl. Acids Res.9:2871-2888). Varying FRT sites are ligated downstream of theterminators as shown in the table. A third expression unit is present inPHP9643 and has an FRT1/GFPm fusion cloned using the flanking NheI siteof FRT1 (SEQ ID NO:2) to remove the ATG start codon of GFPm, therebymaking it non-functional in the existing construct, but where correctexcision of sequences between FRT1 (SEQ ID NO:2) sites can bring theGFPm in frame with the ubiquitin promoter and ATG of the firstexpression unit, thereby making it functional. Downstream of GFPm is thePinII terminator followed by an FRT5 sequence (SEQ ID NO:3).

[0096] PHP9643 was cloned into a pUC derived plasmid backbone. All othervectors were cloned into a pSB11 (See, for example, EPA0672752A1,EPA0604662A1, EPA0687730A1 and U.S. Pat. No. 5,591,616) type plasmidwith the expression units contained between the TDNA border sequences.All are oriented with expression unit one adjacent to the right border.The pSB11-based plasmids were integrated into the super binary plasmidpSB1 (See, for example, EPA0672752A1, EPA0604662A1, EPA0687730A1 andU.S. Pat. No. 5,591,616) by homologous recombination between the twoplasmids. E. coli strain HB101 containing the pSB11 derivatives wasmated with Agrobacterium strain LBA4404 harboring pSB1 to create thecointegrate plasmids PHP10616, PHP11407, PHP11410, PHP11457, PHP11599,PHP11893 and PHP14220 in Agrobacterium (by the method of Ditta et al.(1980) Proc. Natl. Acad. Sci. USA 77:7347-7351). The cointegrates wereverified by Agrobacterium resistance to spectinomycin and SalIrestriction digests.

[0097] Table 2 also includes one example of a vector for creating atarget line where the FRT sites are inserted in the maize ubiquitinintron (last entry) as an alternative location for placement of FRT orother target sites.

[0098] Following selection of stably transformed events, samples ofthese target lines were cryopreserved as a supply for future experimentsusing the approach described by Peterson (see application Ser. No.08/859,313, now U.S. Pat. No. 6,143,563). For several but not allevents, another sample callus from several of the stable transgenicevents was grown, transferred onto regeneration medium to induceplantlet formation and plants were subsequently recovered and grown tomaturity (Register et al. (1994) Plant Mol. Biol. 25:951-961).

EXAMPLE 5 Demonstration of Functionality of Novel FRT Sites in Plants

[0099] (A) Excision of DNA Sequences between Two Identical FRT Sites,but Not when Flanked by Two Non-Identical FRT Sequences

[0100] The extent of intra-plasmid recombination was examined in plantsusing the FRT excision constructs described in Table 3 below. Thevectors PHP10968, PHP10998, PHP10969, PHP11272, PHP11243, PHP11244,PHP12140, PHP12141, PHP12156, and PHP12157 were constructed by ligatingthe maize Ubiquitin promoter upstream of FRT sequences using NcoI orother sites that maintained the ATG start codon. The FRT sequence wasfused in frame at the flanking XhoI site to a GFPm sequence containing aserine to threonine mutation at amino acid residue 65 in the wild typesequence (new sequence termed GFPm-S65T). The pinII terminator wascloned downstream of GFPm. The second expression unit consists of apromoterless FRT, cloned with the 5′ flanking NheI site to remove theATG start codon, fused in frame to the GUS coding sequence (Jefferson etal. (1986) Proc. Natl. Acad. Sci. USA 83: 8447-8451) and followed by thepinII terminator. The vector backbone is a pUC derived plasmid in allcases. Experiments were conducted by bombarding the indicated plasmidsinto maize cells along with construct PHP5096, which carries afunctional expression cassette for FLP protein. PHP5096, the FLPmexpression vector that was used in experiments with the excision andsubstrate vectors, consists of the maize Ubiquitin promoter clonedupstream of the FLPm coding sequence (U.S. patent application Ser. No.08/972,258 to “Novel Nucleic Acid Sequence Encoding FLP Recombinase” nowU.S. Pat. No. 5,929,301) and the pinII terminator in a pUC derivedplasmid backbone. In each case, successful excision would removeintervening sequences between the indicated FRT sites thereby bringingan inactive uidA (GUS) gene in frame with and in proximity to theubiquitin promoter resulting in GUS activity. If excision does notoccur, no GUS expression is expected. The results for GUS expressionfrom these experiments are indicated in Table 4 below. In these studiesefficient excision occurred only where constructs contained twoidentical FRT sites. In the case of the FRT6 (SEQ ID NO:4) and FRT7 (SEQID NO:5) combination, a small amount of recombination was observed,again emphasizing the need for testing target site combinations andjudiciously selecting appropriate combinations for the application.TABLE 3 PHP Upstream-1 Coding-1 Downstream-1 Upstream-2 Coding-2Downstream-2 10968 Ubiquitin ATG/FRT1/GFPm-S65T PinII noATG/FRT1/GUSpinII 10998 Ubiquitin ATG/FRT5/GFPm-S65T PinII noATG/FRT5/GUS pinII11272 Ubiquitin ATG/FRT6/GFPm-S65T PinII noATG/FRT6/GUS pinII 12157Ubiquitin ATG/FRT7/GFPm-S65T PinII noATG/FRT7/GUS pinII 10969 UbiquitinATG/FRT1/GFPm-S65T PinII noATG/FRT5/GUS pinII 11243 UbiquitinATG/FRT1/GFPm-S65T PinII noATG/FRT6/GUS pinII 12140 UbiquitinATG/FRT1/GFPm-S65T PinII noATG/FRT7/GUS pinII 11244 UbiquitinATG/FRT5/GFPm-S65T PinII noATG/FRT6/GUS pinII 12141 UbiquitinATG/FRT5/GFPm-S65T PinII noATG/FRT7/GUS pinII 12156 UbiquitinATG/FRT6/GFPm-S65T PinII noATG/FRT7/GUS pinII 12933 Ubiquitin/FRT1 in 5′UTR GFPm-S65T PinII FRT1 in 5′ UTR/Ubi intron GUS pinII 14076Ubiquitin/FRT1 in intron AHAS PinII FRT1 in Ubi intron GUS pinII 14053Ubiquitin/FRT1 in intron AHAS PinII FRT5 in Ubi intron GUS pinII 14086Ubiquitin/FRT1 in intron AHAS PinII FRT6 in Ubi intron GUS pinII

[0101] TABLE 4 Recombination tested GUS Plasmid between expressionPHP10968 FRT1 and FRT1 +++ PHP10998 FRT5 and FRT5 ++ PHP11272 FRT6 andFRT6 +++ PHP12157 FRT7 and FRT7 +++ PHP9643 FRT1 and FRT5 − PHP11243FRT1 and FRT6 − PHP12140 FRT1 and FRT7 − PHP11244 FRT5 and FRT6 −PHP12141 FRT5 and FRT7 − PHP12156 FRT6 and FRT7 +

[0102] B) Transient Integration of a Second DNA Sequence Flanked by TwoNon-Identical FRT Sequences into Plant Cells

[0103] Summarized in Table 5 below are data from experiments in whichtarget lines created using the plasmids described in Table 2 werebombarded with a substrate plasmid containing a GUS reporter geneflanked by the corresponding FRT sites used in the target constructs.This experiment measured the ability to detect transient GUS expressionshortly after introduction of the substrate plasmid. Since there is nopromoter in front of the first coding sequence in the substrateplasmids, random integration, unless occurring in frame behind anappropriate regulatory sequence elsewhere in the genome, would notresult in GUS expression. This assay system then evaluates the abilityto target FRT-flanked genes into FRT sites in the genome. In general,FRT substrate vectors (Table 6) are constructed as promoterless FRT/genefusions cloned using the 5′ flanking NheI site of the FRT to remove theATG start codon. Genes fused in frame to the FRT with the flanking XhoIsite include one of several scorable or selectable marker genes such asaadA (Svab et al. (1990) Plant Mol. Biol. 14: 197-205), uidA, GFPm,GFPm-C3/intron or bar and are followed by a pinII terminator. In somecases (PHP10259, PHP10603, PHP11561, and PHP11633), plasmids contain asingle expression unit and the second heterologous FRT site is cloneddownstream of the pinII terminator. Substrate plasmids PHP10859,PHP10997, PHP11204, PHP11699, and PHP12190 have in addition to the firstexpression unit described above, a second unit consisting of the maizeubiquitin promoter, the enhanced 35S promoter or a chimeric promoterconsisting of the 35S enhancer region cloned upstream of a syntheticcore promoter termed Rsyn7 (U.S. Pat. No. 6,072,050 which is acontinuation in part of U.S. patent application Ser. No. 08/661,601filed Jun. 11, 1996 now abandoned) cloned upstream of either the HM1,aadA, GUS, or bar coding sequences and the pinII terminator. Aheterologous FRT is inserted downstream of the second terminator.Finally, PHP11003 and PHP11809 contain three expression units. The firstunit is a promoterless noATG/FRT/gene fusion as described above, thesecond unit contains either the chimeric 35S enhancer/Rsyn7 promoterdescribed above or the ZmdJ1 promoter (Baszczynski et al. (1997) Maydica42:189-201) cloned upstream of the GUS coding sequence and the pinIIterminator. The third expression unit consists of the maize ubiquitinpromoter cloned upstream of the HM1 coding sequence, pinII terminatorand a heterologous FRT sequence. All FRT substrate vectors are clonedinto a pUC derived plasmid backbone. Details of the components of thesevectors are described in Table 6. Also listed in Table 6 are two vectorswith alternative placement of FRT sites in the ubiquitin 5′ UTR orintron. TABLE 5 # of GUS PHP9643 PHP11147 PHP11410 PHP11407 PHP11457Spots (n = 74) (n = 127) (n = 32) (n = 38) (n = 113) no spots 17.57%3.15% 6.25% 2.63% 7.96% 1-25 22.97% 48.03% 62.50% 10.53% 27.43% 26-10031.08% 37.80% 18.75% 18.42% 32.74% 101-200 14.86% 8.66% 12.50% 57.89%27.43% too many 13.51% 2.36% 0.00% 10.53% 4.42% to count

[0104] TABLE 6 PHP Coding-1 Downstream-1 Upstream-2 Coding-2 10259NoATG/FRT1/aadA pinII, FRT5 10603 NoATG/FRT1/GUS pinII, FRT5 10859NoATG/FRT1/GFPm PinII Ubiquitin HM1 10997 NoATG/FRT5/GUS PinII UbiquitinaadA 11003 NoATG/FRT1/GFPm PinII E35S/Rsyn7/O′/ADH intron GUS 11204NoATG/FRT1/BAR PinII E35S/Rsyn7/O′/ADH intron GUS 11561 NoATG/FRT6/GUSpinII, FRT1 11633 NoATG/FRT5/GUS pinII, FRT1 11699 NoATG/FRT6/GFPm-C3-PinII Ubiquitin HM1 intron 11809 NoATG/FRT6/GFPm-C3- PinII F3.7 GUSintron 12190 NoATG/FRT1/GUS PinII E35S/35S/O′/ADH intron BARUbiquitin/FRT1 in 5′ UTR HM1 Ubiqutin/FRT1 in intron HM1 PHPDownstream-2 Upstream-3 Coding-3 Downstream-3 10259 10603 10859 pinII,FRT5 10997 pinII, FRT5 11003 pinII Ubiquitin HM1 pinII, FRT5 11204pinII, FRT5 11561 11633 11699 pinII, FRT1 11809 pinII Ubiquitin HM1pinII, FRT1 12190 pinII, FRT5 pinII E35S/35S/O′/ADH intron BAR pinII,FRT5 pinII E35S/35S/O′/ADH intron BAR pinII, FRT5

[0105] Results in Table 5 indicate that the frequency and level of GUSexpression varies among different events, as might be predicted forgenes inserted in different positions in the genome. The prediction isthat once a high frequency, high expressing line is identified, that theexpression of genes subsequently introduced into those same sites willalso be higher than in other lower expressing events.

[0106] C) Stable Integration of a Second DNA Sequence Flanked by TwoNon-Identical FRT Sequences into Plant Cells

[0107] A subset of the stable transgenic “target lines” described inexample 4 above was used in experiments aimed at stably retargeting intothese primary target lines a new gene flanked by the same FRT sites usedin the target lines and cloned in a second construct “substrate”plasmid. Table 7 lists the constructs contained in the primary targetlines (from Table 2), the FRT sites contained in these lines and thesubstrate plasmids (from Table 6) that were subsequently retargeted intothe target lines.

[0108] Table 8 presents data from stable transgenic events whichdemonstrate successful and reproducible targeting of introducedsequences to previously created genomic target sites. The data shown arefor 18 independent target lines, each retargeted with a promoterless GUSconstruct. Since the bar gene was concurrently introduced on the sameplasmid, the proportion of GUS expressing events from the total eventsrecovered on bialophos selection provides a measure of retargetingfrequency relative to random integration. TABLE 7 Target construct FRTsites Substrates being evaluated PHP9643 1/1/5 10603, 10259, 10859,10997, 11003 PHP11147 1/5 10603, 10859, 11003 PHP11407 1/5 10603, 11204,12190 PHP11410 5/1 11633 PHP11457 6/1 11561, 11699, 11809 PHP11893 6/1Experiments in progress

[0109] TABLE 8 # of Random # of Targeted Targeting Target Line EventsEvents Frequency (%) A 13 1 7.1 B 14 1 6.7 C 108 14 11.5 D 18 1 5.3 E 142 12.5 F 9 1 10.0 G 65 1 1.5 H 63 9 12.5 I 71 6 7.8 J 15 1 6.3 K 33 921.4 L 19 2 9.5 M 8 1 11.1 N 12 1 7.7 O 29 4 12.1 P 43 4 8.5 Q 16 3 15.8R 4 1 20.0 S 12 1 7.7 T 10 1 9.1 U 1 2 66.7

EXAMPLE 6 Evaluation of Impact of Introduced FRT Sequences on PlantDevelopment, Gene Expression and Agronomic Performance

[0110] Initial evaluation of the impact of the introduced sequences onplant growth and gene expression is conducted in the greenhouse bymaking regular observations through to pollination and seed set. Plantsare both selfed and crossed to other genotypes to obtain T1 seed forsubsequent greenhouse and field evaluation. For gene expressionevaluation, both qualitative and quantitative data are collected andanalyzed. T1 seeds from transgenic events which give acceptable ordesirable levels of expression and which show no significant negativeimpact on plant development (e.g., have normal developmental morphology,are male and female fertile, etc.) are then grown in managed field plotsalong with non-transgenic control plants, and standard agronomicperformance data is collected and evaluated.

EXAMPLE 7 Conversion of an Introduced Functional FRT Sequence into aSecond Non-Identical Functional FRT Sequence

[0111] The approach taken here to develop a method for convertingbetween different FRT sites for use in various applications is based onthe previously described “chimeraplasty” strategy for making specifictargeted nucleotide modifications at a specified extrachromosomal orgenomic target sequence in animal cells (Yoon et al. (1996) Proc. Natl.Acad. Sci. 93:2071-2076; Cole-Strauss et al. (1996) Science273:1386-1389). This capability in plants, as demonstrated recently inour laboratories and described in WO99/25853, published May 27, 1999, isbeneficial to extending the potential use of the present invention forbroader application. The proposed use of this “chimeraplasty” technologyin the present invention would be to target and modify nucleotides inone FRT site of a pair of non-identical FRT sites flanking a DNAsequence of interest in a way that then makes the two FRT sitesidentical. Subsequent or concurrent expression of FLP recombinase incells with these FRT site modifications would lead to excision of thesequences between these now identical FRT sites, thereby removingspecifically the undesirable DNA sequences from the previously createdstable transgenic event containing those sequences. An application ofthis approach would be for example in the case of a selectable markerwhich is required during initial steps of a breeding or backcrossingprogram to maintain and select for preferred individual plants, butwhich is not desired in the final product.

[0112] A) Vector Design and Construction for Testing Chimeraplasty-BasedFRT Site Conversion

[0113] The target vectors for evaluating this FRT site modificationstrategy are shown generically below, where P1 and P2 represent twodifferent promoters, G1 and G2 represent two genes, and T1 and T2represent two terminator regions; these regions are shown as whiteboxes. Different FRT sites are indicated and shown as dark boxes. Oneversion of the construct incorporates a third unique FRT site downstreamof the second gene and is used to evaluate whether the targetedconversion, in this case, of FRT5 to FRT6 (SEQ ID NO:4), also results inconversion of the downstream FRT1 (SEQ ID NO:2) site to an FRT6 (SEQ IDNO:4) site. In the former case, expression of the downstream gene (G1)should be detected, while if the conversion is not specific to FRT5 (SEQID NO:3) and the FRT1 (SEQ ID NO:2) site is converted also, then bothgene activities will be lost. For the specific examples used here P1 isthe maize ubiquitin promoter, P2 is the enhanced CaMV 35S promoter, G1is the uidA (GUS) gene, G2 is the bar gene, and T1 and T2 are pinIIterminators. It is understood that based on the various descriptions ofvector constructs earlier in this application, a variety of differentpromoters, genes, terminators or DNA sequences or FRT sites could beused in practicing this component method. The DNA cassettes as shownbelow could be assembled into either a pUC-based plasmid for direct DNAdelivery methods (such as particle bombardment) or into a binary vectorfor Agrobacterium-based transformation as described previously. P1 P2 G2T2 G1 T1 FRT6 FRT5 P1 P2 G2 T2 G1 T1 FRT6 FRT5 FRT1

[0114] B) Design of Chimeric Oligonucleotide Molecules forChimeraplasty-Based Targeted Conversion of an FRT Site

[0115] Shown below are specific examples of chimeric molecules thatwould be used to modify a single nucleotide so as to convert the FRT5(SEQ ID NO:3) site to an FRT6 (SEQ ID NO:4) site in constructs asdescribed above. Both the linear sequence of these chimeric molecules aswell as the predicted active form of the molecule (based on the Yoon etal. and Cole-Strauss et al. publications above) are shown. DNA residuesare represented in upper case, RNA residues in lower case, and the siteto be modified (a single nucleotide difference between FRT5, SEQ IDNO:3, and FRT6, SEQ ID NO:4) is underlined and in bold. Two examples ofchimeras are presented below differing in the number of residuesdownstream of the FRT5 (SEQ ID NO:4) site that would be included in thechimeric molecule design and which would thus determine the specificityto the target sequence.

[0116] 1. Chimeric oligonucleotide linear sequence (sequence includessix target-specific residues downstream of the FRT site being modifiedin the target construct and should convert only this single specificFRT5, SEQ ID NO:3, site to an FRT6, SEQ ID NO:4, site) CCTATTCTTCAAAA AGTATAGGAACTTCAGTACTTTTTaguacugaaguuCCTATACTTTuugaagaauaggGCGCGTTTTCGCGC-3′(SEQ ID NO:6)

[0117] Active oligonucleotide conformation of SEQ ID NO:6 TGCGCG--ggauaagaaguuTTTCATATCCuugaagucaugaTT                                            TT                                            T  TCGCGC  CCTATTCTTCAAAA AGTATAGGAACTTCAGTACTT       3′ 5′

[0118] 2. Chimeric oligonucleotide linear sequence (sequence containsresidues specific to only sequences in the FRT site and so shouldconvert any FRT5, SEQ ID NO:3, site in a target molecule to an FRT6, SEQID NO:4, site) (SEQ ID NO:7)5′-TATTCTTCAAAAAGTATAGGAACTTCTTTTgaaguuccuaTACTTTuugaagaauaGCGCGTTTTCGCGC-3′

[0119] Active oligonucleotide conformation of SEQ ID NO:7 TGCGCG--auaagaaguuTTTCATauccuugaagTT                                   TT                                   T TCGCGC  TATTCTTCAAAAAGTATAGGAACTTCT      3′ 5′

[0120] Vector constructions and chimeric oligonucleotide molecules asdescribed above were generated and used in experiments.

[0121] C) Demonstration of Conversion from One FRT Site to Another

[0122] Stable transgenic maize lines are generated with the constructsas described above or with other related ones by transforming in theconstructs and selecting on bialophos as described before. Tissues to beused for chimera delivery are transferred onto non-bialophos-containingmedia and the chimeric oligonucleotides are delivered into cells ofthese stable events by particle bombardment, together with co-deliveryof PHP5096 which carries a functional FLP recombinase expressioncassette. In control experiments, only chimeric molecules or onlyPHP5096 are delivered. After sufficient time for cells to recoverwithout bialophos selection, samples of the bombarded events areevaluated for GUS expression. For those bombarded events containing theconstruct with the downstream FRT1 (SEQ ID NO:2) site which do not showGUS expression, an equivalent sample of cells are plated and grown onmedium with or without bialophos selection to assess sensitivity to thechemical. If the chimeric molecules are specific for modifying only theFRT5 (SEQ ID NO:3) site, then no differences in number and growth ofcells should be observed between treatments with or without selection.Otherwise, reduced growth and recovery should be noted.

[0123] D) Molecular Verification of Stable Conversion of FRT Sites

[0124] DNA from those samples that exhibit GUS expression is isolated,amplified by PCR if necessary, and sequenced by standard methods throughthe region corresponding to the predicted nucleotide conversion. Asufficient stretch of DNA is sequenced to cover the entire originallyintroduced region of DNA so as to confirm correct and specificconversion. Using standard methods for PCR, Southern analysis and/orsequencing of GUS expressing and non-expressing samples establishes thepresence or absence of specific DNA fragments prior to and followingchimeric molecule and FLP recombinase delivery, and thus substantiatesthe visual and biochemical observations made above.

[0125] E) Utility of Chimeraplasty-Based FRT Site Conversion in aTransgene Stacking Strategy for Plants

[0126] Described in FIG. 1 is one potential strategy for combining orstacking multiple desired transgenes at one genomic location using thenon-identical FRT-based system of the present invention. While stackingof genes can be achieved without the use of the targeted FRT conversionmethod described in this example 7, this latter method extends thecapabilities of the system by allowing in vivo conversion of FRT sitesto create new sites, rather than re-introducing new FRT sites bytransformation. In the diagram of FIG. 1, an FRT site with an asteriskbeside it indicates that it was initially created to be non-functionalwith respect to recombination between it and the equivalent FRT sitewithout an asterisk, but which upon conversion with thechimeraplasty-based approach described herein renders it capable ofrecombination with its equivalent non-asterisk counterpart. In thespecific example presented in the figure, this would facilitate forexample removal of a selectable marker either to no longer have itpresent, or to allow one to re-use the selectable marker in futuretransformations. Thus this method also provides a mechanism to recycleselectable markers, as is possible in using the FRT system of thepresent invention alone.

[0127] Discussion

[0128] To date in plants, the major application of the FLP/FRT systemhas been for DNA excision (Lyznik et al. (1993) Nucleic Acids Res.21:969-975). For example, a gene such as a selectable marker flanked byFRT sites is first introduced into plant cells by one of severaltransformation approaches, and stable transgenic events or plants arerecovered via appropriate selection. Then in order to eliminate theselectable marker gene, FLP protein is expressed in the cells eithertransiently by introducing a plasmid carrying a FLP expression cassette,stably following integration of an introduced FLP expression cassette,or by crossing plants carrying the FRT-flanked selectable marker genewith plants carrying sequences for and expressing active FLP protein(WO99/25841, published May 27, 1999, to “Novel Nucleic Acid SequenceEncoding FLP Recombinase”).

[0129] A major problem associated with developing the FLP/FRT system forintegrating genes into animals or plants stems from the fact that therecombination reaction catalyzed by yeast FLP recombinase is areversible process (Sadowski et al. (1995) in Progress in Nucleic AcidResearch and Molecular Biology 51:53-91). For example, followingintroduction of a DNA sequence flanked by similarly oriented FRT sitesinto plant cells in the presence of actively expressing FLP recombinase,recombination should lead to insertion of the new DNA sequences at theendogenous FRT site. However, with continued expression of FLP enzyme,the reverse reaction would lead to re-excision of the introducedsequences because of recombination between the identical FRT sites.Since the reaction is reversible, integration and excision canrepeatedly continue towards equilibrium. As cells divide and the DNAsubstrate concentration per cell decreases, the probability ofintegration decreases, such that in general, as long as active FLPprotein is expressed the reaction will be driven towards thenon-integrated state. To favor integration, a situation must beestablished which precludes re-excision once integration occurs. Anumber of strategies have been suggested, including limiting theduration of activity of FLP recombinase through inducible expression orby directly introducing FLP protein or RNA into cells (Sadowski et al.(1995) Progress on Nucleic Acid Research and Molecular Biology51:53-91), but to date no routine non-random integration system has beenestablished for plants.

[0130] The present invention describes the development of a useful newgene targeting system for plants which utilizes the yeast FLPrecombinase or a modified FLP recombinase designed to work moreefficiently in certain plant species and novel non-identical FRT siteswhich can be used for directional non-reversible DNA integration.Additionally, described herein is a novel use of accessory technologiessuch as “chimeraplasty” permitting in vivo or in vitro modification ofDNA sequences, such as FRT sites to further extend the utility of thesystem. Data provided demonstrate the successful stable integration ofDNA sequences between two previously introduced non-identical FRT sitesin maize. We show also that the DNA sequences between the FRT sites canbe subsequently replaced by a second DNA sequence flanked by the sameFRT sites as the first. Together these results demonstrate that it ispossible to introduce and recover pairs of non-identical FRT sites atcertain genomic locations, that one can select desirable or preferredgenomic locations for expressing DNA sequences of interest, and thatthese selected locations can be used to re-target other DNA sequences ofinterest. Apart from the obvious benefits of being able to integrategenes into the genome of plants, the present invention provides a meansfor facilitating the introduction of novel genes or DNA sequences intogenomic locations previously determined to be particularly beneficialfor gene integration from the perspective of providing suitable levelsof stable expression of the introduced gene(s) and not exhibitingdeleterious impacts on agronomic characteristics including yield. Inaddition the invention provides a system whereby integration of two ormore genes can be targeted to the same genomic location, providing amechanism for “gene stacking”. These stacked genes can then bemaintained and managed as a closely linked pair of traits in breedingprograms.

[0131] Thus this invention also provides an improved method forintroducing, maintaining and breeding multiple genetic traits ofinterest, including agronomic traits, commercially important genes orother heterologous gene products.

[0132] The invention further proposes to use the non-recombinationfeature of non-identical FRT sites to allow creation of a set of‘parental’ lines, which are initially well-characterized for all thedesired expression and performance parameters described above. Theselines then serve as the basis for introduction of new traits into thesame predefined sites in the genome where the initial genes wereintroduced. Many fewer events would need to be generated, sinceintegration would preferentially occur in sites shown to express welland have minimal negative impact on performance.

[0133] All publications and patent applications mentioned in thespecification are indicative of the level of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

[0134] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1 7 1 34 DNA Saccharomyces cerevisiae (14)...(21) spacer region 1gaagttccta ttctctagaa agtataggaa cttc 34 2 69 DNA Unknown (39)...(46)spacer region 2 ccatggctag cgaagttcct attccgaagt tcctattctc tagaaagtataggaacttca 60 gatctcgag 69 3 69 DNA Unknown Description of UnknownOrganismConstructed by synthesizing, annealing and ligatingcomplementary oligonucleotides or by creating primers for PCRamplifications 3 ccatggctag cgaagttcct attccgaagt tcctattctt caaaaggtataggaacttca 60 gtactcgag 69 4 72 DNA Unknown Description of UnknownOrganismConstructed by synthesizing, annealing and ligatingcomplementary oligonucleotides, or by creating primers for PCRamplifications 4 ccatggctag cgaagttcct attccgaagt tcctattctt caaaaagtataggaacttca 60 gacgtcctcg ag 72 5 72 DNA Unknown Description of UnknownOrganismConstructed by synthesizing, annealing and ligatingcomplementary oligonucleotides or by creating primers for PCRamplification 5 ccatggctag cgaagttcct attccgaagt tcctattctt caataagtataggaacttca 60 ctagttctcg ag 72 6 86 DNA Artificial Sequence Descriptionof Combined DNA/RNA Molecule chimeric oligonucleotide 6 cctattcttcaaaaagtata ggaacttcag tactttttag uacugaaguu cctatacttt 60 uugaagaauagggcgcgttt tcgcgc 86 7 70 DNA Artificial Sequence Description ofArtificial Sequence oliognucleotide sequence 7 tattcttcaa aaagtataggaacttctttt gaaguuccua tactttuuga agaauagcgc 60 gttttcgcgc 70

That which is claimed:
 1. A method for locating preferred integrationsites within a genome of a plant cell, said method comprising a)stablyintegrating into the genome of the plant cell a target site comprising afirst nucleotide sequence operably linked to a promoter wherein saidtarget site is flanked by a first recombination site and a secondrecombination site and wherein said first and said second recombinationsites are non-identical; b)locating the preferred integration site byselecting the plant cell expressing said first nucleotide sequence;c)introducing into the plant cell expressing said first nucleotidesequence a transfer cassette, said transfer cassette comprising anucleotide sequence of interest flanked by the first and the secondrecombination sites; and, d)providing a recombinase that recognizes andimplements recombination at the first and the second recombinationsites, wherein said preferred integration site does not disruptexpression of an essential sequence and provides for adequate expressionof said first nucleotide sequence.
 2. The method of claim 1, whereinintroducing comprises sexual breeding.
 3. The method of claim 1, whereinsaid first or said second recombination site is selected from the groupconsisting of a FRT site, a mutant FRT site, a LOX site, and a mutantLOX site.
 4. The method of claim 3, wherein said first or said secondrecombination site is the FRT site and the mutated FRT site.
 5. Themethod of claim 4, wherein said mutant FRT site is FRT 5 (SEQ ID NO:3),FRT 6 (SEQ ID NO:4), or FRT 7 (SEQ ID NO: 5).
 6. The method of claim 1,wherein said plant cell is from a dicot.
 7. The method of claim 1,wherein said plant is from a monocot.
 8. The method of claim 7, whereinsaid monocot is maize.
 9. The method of claim 1, wherein said firstnucleotide sequence is a marker gene.
 10. The method of claim 1, whereinsaid recombinase is FLP.
 11. The method of claim 1, wherein saidrecombinase is Cre.
 12. The method of claim 1, wherein said recombinasehas been synthesized using maize preferred codons.
 13. The method ofclaim 1, wherein locating preferred integration sites comprisesselecting the plant cell expressing said first nucleotide sequence andshowing no significant negative impact on agronomic performance.
 14. Themethod of claim 1, wherein said plant cell is in a plant.
 15. The methodof claim 14, wherein said plant is a monocot.
 16. The method of claim15, wherein said monocot is maize.
 17. The method of claim 14, whereinsaid plant is a dicot.